DETECTION OF GSTP1 METHYLATION IN PROSTATIC SECRETIONS USING COMBINATORIAL MSP ANALYSIS MARK L. GONZALGO, MASASHI NAKAYAMA, SHING M. LEE, ANGELO M. DE MARZO, AND WILLIAM G. NELSON ABSTRACT Objectives. To evaluate the utility of methylation-specific polymerase chain reaction analysis of the pi-class glutathione-S-transferase (GSTP1) gene promoter in prostatic secretions for cancer detection and prognos- tication. Methods. Prostatic secretions were obtained from a total of 100 radical prostatectomy specimens imme- diately after surgical extirpation. GSTP1 promoter methylation was assessed by methylation-specific poly- merase chain reaction analysis using two different primer sets. Correlations between GSTP1 promoter methylation and clinical and pathologic variables were examined. Results. The sensitivity for detection of GSTP1 methylation in prostatic secretions from men with clinically localized prostate cancer using two different primer sets was 76% and 54%. Methylation of the GSTP1 promoter was detected by both primer sets in 44% and by at least one primer set in 86% of the prostatic secretion specimens. The degree of methylation detected in the prostatic secretions was associated with the extent of cancer (predominant involvement of one or both sides of the gland; P = 0.02) and increasing age (P = 0.009). Conclusions. Genomic DNA with GSTP1 promoter methylation can be detected in prostatic secretion specimens from the great majority of men with localized prostate cancer. Assays of GSTP1 promoter methylation in prostatic massage fluid or ejaculate may therefore serve as useful adjuncts to existing methods for prostate cancer screening and prognostication. UROLOGY 63: 414–418, 2004. © 2004 Elsevier Inc. I t is estimated that 220,900 new cases of prostate cancer will have been diagnosed and 28,900 men will have died of the disease in 2003. 1 The most frequent somatic genome alteration appear- ing during prostate cancer development is hyper- methylation of the pi-class glutathione-S-trans- ferase (GSTP1) gene promoter. 2,3 Abnormal methylation of gene promoter regions is a common phenomenon observed in a variety of human can- cers and is associated with transcriptional silenc- ing. 4 Methylation of the GSTP1 promoter is highly specific for the presence of prostatic neopla- sia and is not typically present in normal prostatic tissue. Aberrant GSTP1 methylation has been reported to occur in more than 90% of prostate cancer cases and has been detected in the urine and ejaculate of men affected with the disease. 5–7 Analysis of pros- tatic secretions obtained directly from radical pros- tatectomy specimens permits additional evaluation of the clinical utility of methylation-specific poly- merase chain reaction (MSP) for the detection of methylation changes associated with prostate can- cer in urine or ejaculate. The frequency of GSTP1 methylation in prostatic fluid may represent the maximal sensitivity (ie, best case scenario) for de- tection of this epigenetic alteration. To estimate this sensitivity, we used MSP to detect methylated GSTP1 alleles in DNA from prostatic secretions ob- tained after radical prostatectomy. This work was supported by an Award from the Association for Cure of Cancer of the Prostate (CaPCURE) and by NIH/NCI grants CA58236 and CA70196. W. G. Nelson has a patent (United States patent 5,552,277) titled “Genetic Diagnosis of Prostate Cancer.” From the Departments of Urology and Pathology, James Buchanan Brady Urological Institute and Oncology Center, Johns Hopkins Medical Institutions, Baltimore, Maryland Reprint requests: William G. Nelson, M.D., Ph.D., Sidney Kim- mel Comprehensive Cancer Center, 1650 Orleans Street, CRB 151, Baltimore, MD 21231-1000 Submitted: May 15, 2003, accepted (with revisions): August 29, 2003 BASIC SCIENCE © 2004 ELSEVIER INC. 0090-4295/04/$30.00 414 ALL RIGHTS RESERVED doi:10.1016/j.urology.2003.08.039